The field of the invention is mammalian receptor proteins, and nucleic acids encoding same.
The transforming growth factor betas (TGF-.beta.s) are a family of multifunctional peptide hormones that play important roles in many aspects of cellular function. TGF-.beta. can inhibit the differentiation of certain mesodermal cells, induce differentiation of others, and inhibit proliferation of cells derived from epithelial, endothelial, neuronal, hematopoietic, lymphoid, and fibroblastic origins (Roberts and Sporn, in Peptide Growth Factors and Their Receptors, Sporn and Roberts, Eds., Springer-Verlag, Heidelberg, Germany, 1990, pp. 421-427; Massague, Annu. Rev. Cell Biol. 6:597, 1990). In addition to its effects on individual cells, TGF-.beta. is also important in many biological processes (Roberts and Sporn, supra). For example, TGF-.beta. is an important regulator of immune responses, wound healing, cell adhesion, cell-cell recognition, and extracellular matrix deposition. TGF-.beta. acts as an immune suppressant by counteracting the proliferative effects of interleukin 2 (IL-2) on T and B cells, and by inhibiting the induction of NK cell activities and of LAK cells by IL-2 (Rook et al., J. Immunol. 136:3916, 1986). In addition, TGF-.beta. inhibits T cell-dependent polyclonal antibody production, mixed leukocyte reactions, and the development of CTLs (Ranges et al., J. Exp. Med. 166:991, 1987). Disregulation of TGF-.beta. function is implicated in the pathological processes of several diseases, including arthritis, atherosclerosis, and glomerulonephritis. In addition, cells that lose the ability to respond to TGF-.beta. may be more likely to exhibit uncontrolled proliferation and become tumorigenic. In contrast to these proliferation-inhibiting effects of TGF-.beta., the protein is known to be chemotactic and mitogenic for fibroblasts, and to be a potent chemoattractant for macrophages. It also possesses angiogenic activity.
The biological effects of TGF-.beta. are mediated by several specific cell surface proteins, including the so-called type I, type II, type III, type IV, type V, and type VI TGF-.beta. receptors. Most of these were initially identified by chemically crosslinking radioiodinated TGF-.beta. to cell surface proteins, using the bifunctional reagent DSS. Almost all TGF-.beta.-responsive cell lines express both the type I and type II receptors, although the levels of their expression may vary among different cell types. Neither type I nor type II receptor in the absence of the other can mediate a TGF-.beta. response, although the type II receptor alone is capable of binding TGF-.beta. (Wrana et al., Cell 71:1, 1992; Boyd et al., J. Biol. Chem. 264:2272, 1989). It appears that the ligand signals through a heteromeric complex between the type I and type II receptors (Wrana et al., supra). The type II TGF-.beta. receptor has been cloned; it is a member of the transmembrane serine/threonine receptor kinase family (Lin et al., Cell 68:1, 1992).
M ullerian Inhibiting Substance (MIS) plays a critical role in normal sexual dimorphism as one of the early manifestations of the SRY genetic switch (Gubbay et al., Nature 346:245-250, 1990; Sinclear et al., Nature 346:240-244, 1990; Berta et al., Nature 348:448-350, 1990; Haqq et al., Proc. Natl. Acad. Sci. USA 90:1097-1101, 1993). MIS subsequently causes regression of the M ullerian duct, inhibition of aromatase activity which leads to increased synthesis of testosterone, and probably morphological differentiation of the sex cords as seminiferous tubules, thus assuring the male phenotype. Jost's seminal observations in the late 1940s first defined a "M ullerian Inhibitor" responsible for regression of the M ullerian ducts in the male mammalian embryo (Jost, Arch. Anat. Micro. Morphol. Exp. 36:271-315, 1947). MIS was found to be a 140 kDa protein produced by the Sertoli cell (Blanchard and Josso, Pediatr. Res. 8:968-971, 1974); it was subsequently purified to homogeneity (Budzik et al., Cell 21:909-915, 1980, Cell 34:307-314, 1983; Picard et al., Mol. Cell. Endocrinol. 34:23, 1984), using the bioassay of M ullerian duct regression devised by Picon (Arch. Anat. Microsc. Morphol. Exp. 58:1-19, 1969) as a monitor. The bovine and human genes were cloned (Cate et al., Cell 45:685-698, 1986a) and subsequently expressed and produced in mammalian cell cultures (Cate et al., Cold Spring Harbor Symposium 51:641-647, 1986b; Epstein et al., In Vitro Cellular and Developmental Biol. 25:213-216, 1989); more recently, the rat (Haqq et al., Genomics 12:665-9, 1992) and mouse (Munsterberg and Lovell-Badge, Development 13:613-624, 1991) genes have also been cloned. Overexpression of MIS in transgenic female mice caused regression of M ullerian ducts and seminiferous tubular differentiation (Behringer et al., Nature 345:167-70, 1991). Several patients with Retained M ullerian Duct Syndrome were found to have point mutations in the MIS gene (Knebelman et al., Proc. Natl. Acad. Sci. 88:3767-3771, 1991), which has been localized to the short arm of chromosome 19 (Cohen-Hagenaur et al., Cytogenet. Cell. Genet. 44:2-6, 1987). In mice, the MIS gene is located on chromosome 10 (King et al., Genomics 11:273-283, 1991).
MIS is a member of the large TGF-.beta. family, which includes, besides TGF-.beta. (Derynck et al., Nature 316:701-5, 1985), activin (Ling et al., Nature 321:779-82, 1986; Vale et al., Nature 321:776-779, 1986); inhibin (Mason et al., Nature 318:659-63, 1985); decapentaplegia complex (Padgett et al., Nature 325:81-4, 1987); Vg-1 (Weeks and Melton, Cell 51:861-7, 1987); and bone morphogenesis factors (Wozney et al., Science 242:1528-34, 1988). A common feature of some members of this gene family is that latent precursor can be activated by plasmin cleavage and release of 25 kDa carboxyl terminal dimers.
Although originally defined and named by its ability to cause regression of the M ullerian duct, other functions have emerged for MIS. Its localization to the preantral and smaller antral follicles by immunocytochemical techniques (Takahashi et al., Biol. Reprod. 35:447-53, 1986a; Bezard et al., J. Reprod. Fertil. 80-509-16, 1987; Ueno et al., Endocrinol. 125:1060-1066, 1989a; Ueno et al., Endocrinology 124:1000-1006, 1989b) and its ability to inhibit germinal vesicle breakdown (Takahashi et al., Mol-Cell-Endocrinol. 47:225-34, 1986b; Ueno et al., Endocrinology 123:1652-1659, 1988) led to the hypothesis that it was involved in meiotic inhibition in the ovary. Developmental and experimental correlations support such a function in the testis (Taketo, et al., Devel. Biol. 146:386-395, 1991), where analysis of timing of expression suggests that MIS may be responsible for inhibition of germ cell division. Hutson and Donahoe (Endocrine Reviews 7:270-283, 1986) speculated that MIS may also play role in the transabdominal portion of testicular descent, and Vigier et al. (Development 100:43-55, 1987; Proc. Natl. Acad. Sci. USA 86:3684-8, 1989) have provided evidence that it functions as an inhibitor of aromatase in developing ovaries. Catlin et al. (Am. J. of Obstet. and Gynecol. 159:1299-1303, 1988; Am. Rev. Resp. Dis. 141:466-470, 1990) showed that MIS decreases surfactant accumulation in fetal lungs, thus contributing to the male preponderance in newborn infants of Respiratory Distress Syndrome. The development of a specific serum MIS ELISA (Hudson et al., J. Clin. and Metab. 70:16-22, 1990; Josso et al., J. Clin. Endocrinol. Metab. 70:23-7, 1990) has led to its experimental use as a diagnostic tool for the elucidation of the pathophysiology of ambiguous genitalia in the newborn, and for the use of serum MIS as a marker of granulosa and sex cord tumors in the adult female. Furthermore, the extraordinarily high MIS level observed by Gustafson et al. (New Eng. J. Med. 326:466-71, 1992) in a patient with a sex cord tumor (3200 ng/ml, compared to a normal level of 2-3 ng/ml) provides evidence that MIS is not toxic at these levels.
The role of MIS as a fetal inhibitor has led to the hypothesis that it might act as a tumor inhibitor, particularly of tumors emanating from the M ullerian ducts (Donahoe et al., Science 205:913-915, 1979; Donahoe et al., Ann. Surg. 194:472-480, 1981; Fuller et al., J. Clin. Endocrin. Metab. 54:1051-1055, 1982; Fuller et al., Gynecol. Oncol. 17:124-132, 1984; Fuller et al., Gynecol. Oncol. 22:135-148, 1985). Experimental evidence has accumulated supporting the ability of recombinant human MIS to exert an antiproliferative effect against genital tract tumors in colony inhibition assays, subrenal capsule assays (Chin, et al., Cancer Research, 51:2101-6, 1991), and now metastases assays, and more recent evidence has shown an antiproliferative effect against a series of human ocular melanomas (Parry et al., Cancer Research 51:1182-6, 1992). MIS has been shown to block tyrosine autophosphorylation of EGF receptors (Coughlin et al., Mol. and Cell. Endocrin. 49:75-86, 1987; Cigarroa et al., Growth Factors 1:179-191, 1989).
Inhibin, another member of the TGF-beta family described above, is primarily secreted by Sertoli and granulosa cells of the male and female gonad. This nonsteroidal regulatory hormone, first described in 1932 (McCullagh, Science 76:19-20), acts specifically to inhibit FSH release from the pituitary (Vale et al., Recent Prog. Horm. Res. 44:1-34, 1988). Biologically active inhibin, however, was not purified and characterized well until the successful cloning of its genes in 1985-86 (Mason et al., Nature 318:659, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091, 1986; Mayo et al., Proc. Natl. Acad. Sci. USA 83:5849, 1986; Esch et al., Mol. Endocrinol. 1:388, 1987). Inhibin was shown at that time to be a glycoprotein heterodimer composed of an alpha-chain and one of two distinct beta-chains (beta-A, beta-B) (Mason et al., Biochem. Biophys. Res. Comun. 135:957, 1986). The alpha chain is processed from an initial species of 57 kDa to form an 18 kDa carboxyl-terminal peptide, while the mature beta chain of 14 kDa is cleaved from the carboxyl-terminus of a 62 kDa precursor, which would then account for the biologically active 32 kDa species which predominates in serum (DeKretser and Robertson, Biol. Reprod. 40:3347, 1989). Many other forms of bioactive inhibin with MS's of 32-120 kDa, however, have been isolated as well (Miyamoto et al., Biochem. Biophys. Res. Commun. 136:1103-9, 1986). In addition, beta-chain dimers (beta-A/beta-A, beta-B/beta-B, or beta-A/beta-B) which selectively stimulate FSH secretion from the pituitary have been identified and are called activin A, activin B, and activin AB, respectively (Vale et al., Nature 321:776, 1986; Ling et al., Nature 321:779, 1986).
As is the case with MIS, many additional functions have been postulated for inhibin and its subunits besides FSH regulation. Inhibin alpha, beta-A, and beta-B subunit RNAs have been shown to be expressed in a variety of rat tissues, including the testis, ovary, placenta, pituitary, adrenal gland, bone marrow, kidney, spinal cord, and brain (Meunier et al., Proc. Natl. Acad. Sci. USA 85:247-51, 1988). The pattern of testicular inhibin secretion appears to be developmentally regulated. In the rat, inhibin increases during maturation until 30-40 days after birth, after which values rapidly return to juvenile levels (Au et al., Biol. Reprod. 35:37, 1986). Inhibin subunits also seem to have a paracrine effect on Leydig and theca interna cell androgen synthesis (Hsueh et al., Proc. Natl. acad. Sci. USA 84:5082-6, 1987). Many studies have demonstrated the changes in inhibin which occur throughout the estrus cycle, and therefore, its role in modulating FSH in adult females (Hasegawa et al., J. Endocrinology 121:91-100, 1989; McLachlan et al., J. Clin. Endo. Metab. 65:954-61, 1987). Furthermore, changes in local inhibin concentrations may be involved in the regulation of ovarian folliculogenesis (Woodruff et al., Science 239:1296-9, 1988; Woodruff et al., Endocrinology 127:3196-205, 1990). Bioactive inhibin has been shown to be produced by human placental cells in culture and to be involved in a short-loop feedback between gonadotropin-releasing hormone and human chorionic gonadotropin (Petraglia et al., Science 237:187-9, 1987). Finally, a number of patients with ovarian granulosa cell tumors have been described who had markedly elevated serum inhibin levels secondary to tumor production of this hormone (Lappohn et al., NEJM 321:790-3, 1989).
Most of the data that exist concerning serum inhibin levels in humans has been obtained using a heterologous radioimmunoassay comprised of a polyclonal antibody to purified, intact bovine inhibin and radiolabeled 32 kDa bovine inhibin (McLachlan et al., Mol. Cell. Endocrinol. 46:175-85, 1986). Such studies have evaluated normal cycling females and adult males (McLachlan et al., J. Clin. Endo. Metab. 65:954-61, 1987; McLachlan et al., J. Clin. Invest. 82:880-4, 1988), pubertal males (Burger et al., J. Clin. Endo. Metab. 67:689-694, 1988), normal pregnant women (Abe et al., J. Clin. Endocrinol. Metab. 71:133-7, 1990), and a variety of reproductive disorders (Scheckter et al., J. Clin. Endocrinol. Metab. 67:1221-4, 1988; DeKretser et al., J. Endocrinol. 120:517-23, 1989). However, recent work has shown that this assay detects inhibin alpha-subunits as well as intact dimeric hormone, and, therefore, these results should be interpreted with caution (Schneyer et al., J. Clin. Endocrinol. Metab. 70:1208-12, 1990).